Scanner photometer methods

ABSTRACT

A scanning photometer and attendant methods are provided. The scanning photometer is generally characterized by first and second fluorophore excitation sources, an objective lens, and a common emission detector for the detection of first and second fluorophore emission originating from the excitation of the fluorophores via passage of excitation energy, via an optical path of the objective lens, from the excitation sources. Excitation energy and emission energy conditioning elements are like-wise provided, operatively interposed before or after the objective lens as the case may be.

This is an international patent application filed under 35 U.S.C. §363claiming priority under 35 U.S.C. §119(e) (1) to U.S. provisional pat.appl. ser. No. 61/104,109, filed Oct. 9, 2008 and captioned “ScannerPhotometer and Methods,” said application incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention generally relates to the field of optics, moreparticularly, to instruments and/or methods for detecting and/ormeasuring light intensity or optical properties of solutions orsurfaces, and more particularly still, to a scanner photometer andmethods having particular utility with regard to fluorescence detectionand/or quantification.

BACKGROUND OF THE INVENTION

The minimization of uncertainty with regard to data is universally heldas advantageous. Moreover, and fundamentally, in as much as there existsa premium on precision in acquiring data, to appreciate and acknowledgeuncertainty with regard to data acquisition is likewise of value so that“meaningful” data informs the technician, clinician, scientist,engineer, etc.

Fluorescence photometry is premised upon the adsorption and subsequentre-radiation of light, i.e., electromagnetic radiation, by organic andinorganic specimens. Via fluorescence labeling or tagging of a specimen,sample, etc. with a fluorophore (a/k/a, a fluorochrome), i.e., afunctional group of a molecule which absorbs energy of a specificwavelength and re-emits energy at a different, but equally specificwavelength wherein the amount and wavelength of the emitted energydepend on both the fluorophore and the chemical environment of thefluorophore; the application of excitation energy to such specimen; and,monitoring or sensing of emission energy from the excited specimen, thepresence/absence and/or quantity of a tagged component of the specimenmay be obtained or ascertained. Well known illustrative, non-limitingfluorescence photometry fields include, molecular biology, biochemistry,and pharmaceutical science, with particular, but hardly exclusiveutility known with regard to genotyping, sequencing, screening andclinicals.

Characteristic of scanning operations is a scanner, a target, and motionof one element with respect to the other. Upon reflection, it should beappreciated that potential scanning issues arise in connection to theinherent motion of scanning, e.g., getting either the scanner or thetarget from point A to point B, as well as the relative positioning oralignment from one target to the next for the purpose of dataacquisition.

Optical scanning heads, more particularly photometers, includecomponents, such as photomultiplier tubes (PMTs), which are susceptibleto mechanical vibration. One cause of mechanical vibration is thephysical quantity known as “jerk” (J), mathematically the firsttime-derivative of acceleration (A), i.e., J=dA/dt, which is manifest orinherent in the motion cycle of a scanning carriage to which a scanningphotometer is commonly attached. In an effort to eliminate jerk relatedvibration in or with respect to the photometer, it has heretofore beenfound advantageous to provide a static or stationary photometer, and adynamic specimen medium, e.g., a selectively positionable plate, whichpasses thereby. With increased processing throughput via the use ofcontinuously spooled array tape and the like, use of a stationaryphotometer has become impractical, however, any inconsistency in therelative position between the scanning photometer and each target of aseries of targets, due to any reason, can cause significant differencesin the quality of output data.

In the context of a multi-dye fluorescence assessment, thecharacteristic multiple fluorophore excitation energy directed to afirst target, and returned fluorophore emission energy from the excitedtarget, e.g., the paths associated therewith, must be precise, certainand repeatable/reliable in relation to the target itself, as well asfrom a first target to successive targets. Repeatability of dataacquisition conditions is highly valued, and in the context of reducedspecimen/target volumes for scanning, and with regard to the high volumethroughput available with array tapes, repeatability, reliability andprecision is an increasing challenge. Thus, in light of at least theforgoing, it remains advantageous to provide a scanning photometercapable of precise, reliable and repeatable data acquisition, moreparticularly, such scanning photometer and associated methods whichcomplement heretofore known target media processing advancements.

SUMMARY OF THE INVENTION

A scanning photometer and attendant methods are provided. The scanningphotometer is generally characterized by first and second fluorophoreexcitation sources, an objective lens, and a common emission detectorfor the detection of first and second fluorophore emissions originatingfrom the excitation of the fluorophores via passage of excitationenergy, via an optical path of the objective lens, from the excitationsources. Excitation energy and emission energy conditioning elements arelikewise provided, operatively interposed before or after the objectivelens as the case may be.

The scanning photometer is advantageously operatively supported by ascanning rail carriage for reversible travel. More particularly, travelcharacterized by a constant velocity during active scanning of targetsof an array of targets. Moreover, the scanning photometer may furtheradvantageously include a third fluorophore excitation source, excitationenergy therefrom passing through the objective lens, and a furtheremission detector for the detection of fluorophore emissions originatingfrom excitation of the third fluorophore.

In connection to photometric scanning methods, those of the instantinvention may be fairly and generally characterized by the steps ofpassing an objective lens across a series of spaced apart targets, andtransmitting energy from at least two fluorophore excitation sourcesthrough an optical path of the objective lens for receipt by a target ofa series of spaced apart targets in furtherance of exciting at least twofluorophores of the target. Advantageously, but not necessarily, theobjective lens is passed across the series of spaced apart targets at aconstant velocity. Moreover, during a “pass,” energy transmissionsdirected to the target from the fluorophore excitation sources may besimultaneous, periodic, alternated (i.e., transmission from a firstsource; transmission from a second source upon termination of the firstsource transmission; and, a return to first source transmission upontermination of the second source transmission), or pulsed (i.e.,transmission from one source of the two sources is pulsed during theenergy transmission of another of the two sources).

Broadly, and as will be later detailed, there are provided preferredoptical operational modalities for a multi-dye scanning photometer,namely, multi-dye measurements characterized by notions of“colocalization” (i.e., obtaining independent measurementssimultaneously at a common localized alignment), “consensing” (i.e., useof a single emission detector for the detection of two fluorophoreemissions), “consituational” (i.e., the elimination or minimization ofdifferent situational relationships or inherent conditions for thespecimen between measurements), and “confocal” (i.e., establishing acommon local alignment for and/or between the specimen focal point andthe aperture focal point).

More specific features and advantages obtained in view of those featureswill become apparent with reference to the drawing figures and DETAILEDDESCRIPTION OF THE INVENTION.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts, in perspective view, a scanning photometer apparatuscharacterized by a scanning photometer in accord with a preferredteaching of the instant disclosure, a scanning module door omitted toshow underlaying structures;

FIG. 2 depicts, in perspective view with portions omitted for the sakeof clarity, elements of the scanning photometer apparatus of FIG. 1 andtheir interrelationships as viewed from the vantage point of the unwindmodule;

FIG. 3 depicts, in perspective view, operative elements of the scanningphotometer of FIG. 1;

FIG. 4 depicts, in exploded view, select elements of the scanningphotometer of FIG. 3; and,

FIG. 5 represents a motion profile, more particularly, an accelerationprofile of the scanning photometer of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

The following description immediately proceeds with general reference toFIGS. 1 & 2, wherein a preferred, non-limiting scanning photometerapparatus is generally depicted, and thereafter with particularreference to FIGS. 3 & 4 wherein an optical reader or optical readerhead of the subject apparatus is depicted. Finally, reference is made toFIG. 5 with regard to preferred apparatus operation.

Contextually, the apparatus of FIGS. 1 & 2 is advantageously, but notexclusively suited for high speed scanning, derived, at least in part,from the utilization of array tape (see e.g., U.S. Pat. No. 6,878,345B1) and an automated system/process for/of handling same (see e.g., theNexar™ array tape automation instrument by Douglas Scientific,Minnesota, USA). Moreover, while the subassembly of FIGS. 3 & 4 may begenerally and fairly characterized as an optical reader head, it may bemore particularly and fairly characterized as a multi-dye fluorescencephotometer, having particular utility with regard to, for example andwithout limitation, single nucleotide polymorphisms (SNP) genotyping viaallele specific polymerase chain reaction (PCR) assays (see e.g., U.S.Pat. Nos. 6,537,752 B1 and 6,632,653 B1) as well as emerging relatedprocesses, see e.g., Applicant's copending international patentapplication ser. no. PCT/US09/42007 entitled “High Throughput ScreeningEmploying Combination of Dispensing Well Plate and Array Tape.”

With reference now to FIGS. 1 & 2, a scanner photometer apparatus 100according to the preferred teachings of the present invention is shown,and generally includes an unwind module 70, a scanning module 80, and arewind module 90. In the unwind module 70, the spool holder 71 holdstape 92, in spool form, which is characterized by an array of “wells”(i.e., rows and columns of spaced apart aqueous volumes set across thelength and width of the tape respectively). The tape 92 feeds off spoolholder 71 into the tape guide 72, and onto a tape drive 73 which conveysthe tape 92 toward and through the scanning module 80. The tape 92 isindexingly conveyed via tape drive 73 so as to stop for scanning by theoptical reader head 10, more particularly, column scanning as depicted.

A bar code reader/reading station 14 (FIG. 2), characterized by, amongother things, a bar code hold down 89, is advantageously supplied fordetecting and “reading” sample/well indicia associated with the conveyedtape or tape segment in furtherance of correlating a select photometricscanning result with a select target sample (i.e., target well). Lightfrom the bar code reader of the reading station is generally shieldedfrom photometric scanning operations by at least a portion of the barcode reading station, e.g., the bar code hold down 89.

The tape is generally held flat in tape scanning region 85 by a tapehold down 86, with light from the region advantageously shielded by darkenclosure 84 (FIG. 4). Moreover, the tape hold down acts as a funnelguide for the ends of the tape, and further shields bar code scanningemissions from photometric scanning operations of the scanning module.Tape hold down 86, characterized by apertures 87 configured so as tocorrespond or register with well locations of the conveyed tape, isadvantageously, but not necessarily, comprised of a resilient member orelement, or is otherwise adapted so as to automatically compensate foror respond to tape thickness variations while nonetheless maintaining asurface of the tape in operative engagement with groove plate 83.

With regard to the scanning module 80, a movable carriage 82, to or uponwhich the optical reader head 10 is operatively mounted, is translatableupon a carriage rail 30 or the like such that the optical reader headpasses through tape scanning region 85. The optical reader head isadvantageously designed to be easily connected with the scanning railcarriage. As should be readily appreciated with reference to thefigures, the motion of the carriage, and thus the optical reader head,is perpendicular to the tape drive motion, and parallel to the topsurface or face of groove plate 83.

Functionally, the optical reader head 10 moves in a first direction asit scans each well in the target column. Once the target column isscanned, the tape 92 indexes ahead one column, thereby establishing anew target column, and the optical reader head 10 moves in a seconddirection as it scans each well in the subsequent target column, withthe feeding/indexing and scanning process repeating. As the tape 92indexes, it winds on the rewind or uptake spool 91.

In lieu of tape processing via a tape drive, plate processing is enabledvia a plate drive. Plate 81, likewise characterized by an array ofwells, can be manually loaded or conveyed into the scanning module 80,more particularly, provided to or within a plate scanning region 88which is oriented with respect to tape scanning region 85 so as to bein-line therewith. Plate motion is generally parallel to the motion ofthe tape, with a top or upper surface of the plate generally parallel toan upper or top surface of the tape. Similar to tape processing, theoptical reader head 10 scans a target column of the plate 81 in a firstdirection, and, thereafter, and subsequent to manipulation of the plate81 as by manual indexing forward one column, the optical reader head 10then reads the new target column in a second direction, with the processrepeating.

With reference now to FIG. 3, and the particulars of FIG. 4, there isdepicted multiple excitation sources, with attendant excitation energyconditioning elements; dual emission detectors, with attendant emissionenergy conditioning elements; and, an objective lens characteristic ofthe optical scanning head, more particularly, the multi-dye fluorescencephotometer of the instant invention. The photometer of FIG. 3, and asbest seen in connection to FIG. 4, advantageously, but not necessarily,includes first 21, second 22 and third 23 fluorophore excitationsources, an objective lens 15, characterized by an optical path 16,through which excitation energy from the fluorophore excitation sourcespass, and, first 61 and second 63 emission detectors for selectivelydetecting fluorophore emissions originating from excitation offluorophores of a target. Advantageously, detection of greater than onefluorophore emission by a single emission detector provides improvedscanning precision and an accompanying upgrade with regard to dataquality.

Excitation energy conditioning elements are operatively interposedbetween the excitation sources and the objective lens.

More particularly, excitation energy conditioning elements comprise, inselective combination, excitation filters and beam splitters for selectpassage of excitation energy to and along the optical path of theobjective lens.

As shown, and to facilitate subsequent discussion, excitation energyfilters 31, 32, and 33 correspond to first 21, second 22 and third 23fluorophore excitation sources. With regard to the excitation energybeam splitters, splitters 40, 41, 42 & 43 are provided for conditioningexcitation energy from fluorophore excitation sources 21, 22, & 23. Aswill be subsequently addressed in connection to emission energyconditioning, excitation energy beam splitters 41 & 43 are arranged soas to likewise selectively condition fluorophore emissions.

Emission energy conditioning elements are likewise operativelyinterposed between the objective lens and the emission detectors. Moreparticularly, emission energy conditioning elements comprise, inselective combination, beam splitters, dedicated and shared, andemission filters for select passage of emission energy from the opticalpath of the objective lens and to the emission detector.

As shown, emission energy beam splitter 42 is provided, and is generallycorrelated with/to first emission detector 61, with splitters 41 & 43conditioning emission energy directed toward second emission detector63, and splitter 42 likewise influencing same. With regard to emissionenergy filters, filters 51 & 53 correspond to first 61 and second 63emission detectors respectively.

The excitation filters of the photometer, which advantageously compriseband pass interference filters, are generally characterized as follows:the design bandwidth of filter 31 is preferably shorter than beamsplitter 40; the design bandwidth of filter 32 is preferably longer thanbeam splitter 40 and shorter than beam splitter 41; and, the designbandwidth of filter 33 is preferably shorter than beam splitter 43. Theemission filters, likewise advantageously comprised of band passinterference filters, are generally characterized as follows: the designbandwidth of filter 51 is preferably longer than beam splitter 41, andshorter than beam splitter 42; and, the design bandwidth of filter 53 ispreferably longer than beam splitter 43.

The excitation and emission filters must provide strong mutual blocking,where: filter 31 is designed to strongly block filters 51 and 53; filter32 is designed to strongly block filters 51 and 53; filter 33 isdesigned to strongly block filters 51 and 53; filter 51 is designed tostrongly block filters 31, 32, and 33; and, filter 53 is designed tostrongly block filters 31, 32, and 33. The excitation and emissionfilter ranges are based upon the spectrum of each fluorophore sought fordetection. Preferably the excitation and emission spectral peakscoincide with the bandwidth of the filters.

The excitation beam splitters of the photometer, which advantageouslycomprise long pass dichroic mirrors, are generally characterized asfollows: the design wavelength of beam splitter 41 is longer than beamsplitter 40; and, the design wavelength of beam splitter 42 is longerthan beam splitter 41; and, the design wavelength of beam splitter 43 islonger than beam splitter 42.

In light of the foregoing, and with particular reference to FIG. 4, anoutline of optical paths associated with the multi-dye fluorescencephotometer follows. Generally, fluorophore excitation paths begin at thefluorophore excitation sources and end at the target, with fluorophoreemission paths commencing at the target and terminating at the emissiondetector, more particularly:

-   -   As to the first fluorophore, a first excitation energy is        emitted from first fluorophore excitation source 21, through        excitation filter 31 for reflection by beam splitter 40 and        further reflection by beam splitter 41 so as to thereafter pass        through objective lens 15 and on to sample 12 whereupon the        first fluorophore emits emission energy back through objective        lens 15, through beam splitter 41 for reflection by beam        splitter 42, through emission filter 51 and to detector or        sensor 61;    -   As to the second fluorophore, a second excitation energy is        emitted from second fluorophore excitation source 22 through        excitation filter 32 and beam splitter 40 for reflection by beam        splitter 41 through objective lens 15, and to sample 12        whereupon the second fluorophore emits emission energy back        through objective lens 15, through beam splitter 41 for        reflection by beam splitter 42, through emission filter 51 and        to detector or sensor 61;    -   As to the third fluorophore, a third excitation energy is        emitted from third fluorophore excitation source 23 through        excitation filter 33 for reflection by beam splitter 43 and        passage through beam splitter 42 and beam splitter 41 through        objective lens 15, and to sample 12 whereupon the third        fluorophore emits emission energy back through objective lens        15, through beam splitters 41, 42, & 43, through emission filter        53, and to detector or sensor 63.

Prior to a presentation of the scanning process, one or more operationalmodalities of the apparatus, and/or methods of the subject disclosure,several concluding particulars with regard to select elements orstructures of the multi-dye fluorescence photometer are warranted.Specifics as to the fluorophore excitation sources, the filters, thebeam splitters, the emission detectors, and the objective lens follows,with a concluding identification of select attendant structures of thefluorophore excitation sources and the beam splitters.

First, in connection to the fluorophore excitation sources, first secondand third sources are advantageously comprised of light emitting diodes(LEDs), more particularly designated, for example and withoutlimitation, as follows: first fluorophore excitation source 21 comprisesa 505 nm center wavelength FAM excitation lamp, e.g., part no. OSRAM LVW5SN-JXKZ-25-S-Z from OSRAM GmbH, Germany; second fluorophore excitationsource 22 comprises a 528 nm center wavelength VIC excitation lamp,e.g., OSRAM LT W5SN-JYKZ-36-Z from OSRAM GmbH, Germany; and, thirdfluorophore excitation source 23 comprises a 590 nm center wavelengthROX excitation lamp, e.g., part no. OSRAM LY W5SN-JYKY-46-Z from OSRAMGmbH, Germany.

Second, in connection to the band pass interference filters, thefollowing specifications are noted for each of excitation filters 31, 32and 33 respectively: center wavelength 485, with a bandwidth of 20(i.e., 485/20); 520/10; and, 590/20. With regard to the emissionfilters, namely emission filters 51 & 53, specifications therefore are550/20 and 620/20 respectively.

Third, with regard to the beam splitters, the following specificationsare noted for each of beam splitters 40, 41, 42 and 43 respectively:center wavelength 510, long pass (i.e., 510LP); 535LP; 570LP; and,605LP.

Fourth, with regard to the emission detectors, first and second emissiondetectors are advantageously comprised of photomultiplier tubes (PMTS),more particularly designated, for example without limitation, the H5784series of metal package PMT products of Hamamatsu Photonics K.K., Japan.

Fifth, with regard to objective lens 15, a Plan Fluor series lens of theCFI60 family of “objectives for biological microscopes” from NikonInstruments, Inc., New York, U.S.A. is particularly well suited forpassage of the excitation and emission energy in the context of thepreviously described components.

Lastly, and with continued reference to FIGS. 4 & 5 as the circumstancewarrants, in as much as the fluorophore excitation sources generallycomprise a source of excitation energy in the form of an LED or thelike, a housing 24 for containing and directing the excitation energy,and a heat sink 25 operatively linked to the LED is provided. Moreover,in as much as the beam splitters generally comprise long pass dichroicmirrors (FIG. 4), a beam splitter body 45, more particularly a two partbody as shown, and an optical beam dump structure 46 are likewiseprovided.

Recalling threshold issues of running the scanner through its paces,e.g., moving from point A to B and back to A, and the ever present needto gather data during the journey, operational modalities with regard tothe subject scanning photometer scanning apparatus, and, as the case maybe, the subject multi-dye fluorescence photometer are generally directedto the minimization of uncertainty with regard to data/informationgathered by the scanning operation. The presentation of a preferredprocess of directing the scanning photometer in relation to the arrayedtargets precedes a presentation of operational steps with regard tooptical operational modalities for the multi-dye fluorescencephotometer.

In the preferred embodiment, the scanning cycle is composed of twomoves: the carriage first moves a given distance in a first direction,and then moves in the reverse direction, back to the starting position,whereupon the cycle repeats. As previously noted, in the context of theforegoing description and semantic, a select column of arrayed targetsis scanned in the first direction, and a column of targets adjacent theselect column is scanned in the reverse direction. Each target of thetargets of the column is read during the zero acceleration (i.e.,constant velocity) portion of the motion profile.

The key to having a minimum jerk move is to avoid torquediscontinuities, with the key to minimization of the magnitude of thejerk being to maintain the torque from the end of the forward move (F)to the start of the reverse move (R). In the light of the instantteaching, the direction change is not accompanied by the customary forcechange to the motor shaft. The force on the shaft never goes to zeroduring direction change between F and R, see e.g., the motion (i.e.,acceleration) profile of FIG. 5 corresponding to the scanning photometerof the instant invention. Jerk assessment parameters comprise thefollowing: (1) the constant velocity of the carriage at which the targetis read; (2) the average acceleration; (3) the average deceleration;and, (4) the shape of the triangular acceleration curve.

The carriage according to the teaching of the present invention can nowbe operated at significantly higher speeds with minimum jerk, and thecorresponding or resulting reduction of vibration. It is to be furthernoted that the profile of FIG. 5 also lends itself to other curves orshapes such as sinusoidal functions.

Thus, the motion profile according to the teaching of the presentinvention reduces vibration due to high-jerk acceleration anddeceleration in the optical reader as it moves across the target. Themotion profile allows the carriage that carries the optical reader toscan the target at high velocities, while minimizing jerk. Moreover,making the system mechanically rigid to minimize deflections, andchoosing a high-resolution servo mechanism to allow accurate positioningaid in greatly reducing the drawbacks of a moveable optical reader.

Turning now to the preferred optical operational modalities for themulti-dye fluorescence photometer, multi-dye measurements characterizedby notions of colocalization, consensing, consituational, and confocalare noted. A brief summary of each follows.

An advantage according to the preferred teaching of the presentinvention is “colocalized (i.e., the same location) multi-dyemeasurement.” A primary goal of the scanner is to measure an accurateratio of multiple fluorophores present within the sample or target. Itis of secondary importance to measure the absolute quantity of eachfluorophore within the sample. The relative spatial intersection of thephotometer optical path with the sample to be measured can have asignificant influence on both the excitation energy imparted into thesample, and the collection of the resulting emission. Therefore, thealignment between multiple fluorophore excitations and emissions has aconsiderable influence on the measured signal strength. One solutiondirected to this issue is to combine multiple excitation and emissionoptical paths through a common objective lens. With this approach,independent measurements may be taken simultaneously at a commonlocalized alignment, or in very rapid succession by flashing theexcitation lamps, effectively at one alignment.

A further advantage according to the preferred teaching of the presentinvention is “consensory (i.e., cosensing using the same sensor)contemporal (i.e., at the same time) multi-dye measurement.” A primarygoal of the scanner is to measure an accurate ratio between first andsecond select fluorophore emissions. As emission sensors may exhibit aunique signal drift, warm-up, or other non-linear behavior, unwelcomeuncertainty is inherent in the dedicated detection of first and secondselect fluorophore emissions with first and second emission detectors.By using a single sensor to measure both first and second selectfluorophore emissions, a more accurate ratio is possible.

In the current state of the art, a single sensor is used (i.e., thenotion of consensory) via scanning an entire array to measure emissionsfrom a first fluorophore, then, subsequent to switching optical filterelements, another repeat scan of the array is conducted. With thisapproach, however, the drift of the sensor will not be the same betweenpasses. This difference will introduce an error in the ratio between thereadings for each sample. In contrast, by quickly “switching” theexcitation lamps, the sensor drift will effectively be the same value atthat moment (i.e., consensory plus contemporal). When the same error isapplied to two measurements, a more accurate ratio is provided. Byquickly switching excitation sources, both fluorophore emissions aredetected/measured independently by the same/common sensor, and at veryshort (i.e., not meaningful) time intervals. This approach providesmeasurements that effectively cancel out the sensor drift.

Yet a further advantage according to the preferred teaching of thepresent invention is “consituational (i.e., the same situation orcircumstance of the target/specimen/sample) multi-dye measurement.” Inthe current state of the art, the samples are moved while the photometerremains stationary to perform a first measurement. As should be readilyappreciated, the sample is plainly at risk of being in a differentsituational relationship or inherent condition between measurements,i.e., at measurement “1” the sample is in a first situation, and atmeasurement “2,” the sample is at a second situation, wherein the firstand second situations are not interchangeable equivalents. Moreover, itis to be appreciated that there may exist scenarios where the specimen,advantageously residing in a sub-microliter liquid reaction volume(i.e., a well) of the array media, undergoes time dependent orindependent changes, physical or otherwise. For example, and withoutlimitation, there is the potential for the fluorophore to float aboutfreely within a well during the intervening time between scans. With thecurrent approach, the samples remain stationary while all themeasurements are performed in very rapid succession, effectively at thesame physical situation or circumstance.

Still further, an advantage according to the preferred teaching of thepresent invention is “confocal (i.e., the same focus) multi-dyemeasurement.” By using an aperture in the emission energy path,scattered light emitted by the sample can be rejected, and only thelight emitted at the focal point of the objective lens can be collectedby the sensor. This approach represents a stronger and/or more preciseform of colocalized measurement. Multiple individual apertures can beused for each sensor, or a common single aperture may be placed in thecombined beam paths.

With regard to preferred, non-limiting method or process particularswhich follow, namely, photometric scanning methods, it is to beunderstood that a preferred scanning context is array scanning. Whetherthe arrayed targets are embodied in an array tape or a plate,characteristic of the media carrying, holding, retaining, etc. theobjects for scanning is a series of spaced apart targets, moreparticularly, a series of spaced apart target columns. Thus, both tapescanning, via a tape scanning region, and plate scanning, via a platescanning region, alone or in combination, are contemplated and believedadvantageous. Moreover, and consistent with the subject disclosure, itis to be understood that scanning entails moving an opticalreader/reader head, more particularly, a photometer, in relation to thearrayed targets, arrayed targets which likewise may be indexed (i.e.,selectively advanced), either from one column to the next, or some otherselect increment subsequent to an initial pass of the photometer.

Photometric scanning methods of the instant invention may be fairly andgenerally characterized by the steps of passing an objective lens acrossa series of spaced apart targets, and transmitting energy from at leasttwo fluorophore excitation sources through an optical path of theobjective lens for receipt by a target of the series of spaced aparttargets in furtherance of exciting at least two fluorophores of thetarget. Advantageously, but not necessarily, the objective lens ispassed across the series of spaced apart targets at a constant velocity.Moreover, during a “pass,” energy transmissions directed to the targetfrom the fluorophore excitation sources may be simultaneous, periodic,alternated (i.e., transmission from a first source, transmission from asecond source upon termination of the first source transmission, and areturn to first source transmission upon termination of the secondsource transmission), or pulsed (i.e., transmission from one source ofthe two sources is pulsed during the energy transmission of another ofthe two sources).

As previously noted, a further operative advantage is obtained viacosensing. More particularly, a preferred photometric scanning methodfurther includes or is characterized by the detection, via a singleemission detector, of two fluorophore emissions corresponding to theexcitation of the two fluorophores of the target. To the extent thatgreater than two emission energies are to be detected, e.g., three as ischaracteristic of allele specific PCR assays, steps associated with afurther excitation energy source and corresponding emission detector arelikewise contemplated.

Lastly, with regard to or even apart from the aforedescribed scanningphotometer apparatus, and subassemblies therefore, optical operationalmodalities, and photometric scanning methodologies, furthermodifications or departures are to be noted. For example, the opticalreader/read head may advantageously include more than one photometer. Ina read head so configured, each photometer may be aimed or otherwisedirected towards different targets or target groups/subsets. Moreover,the photometers may be operatively aligned with regard to the samecolumn of the arrayed targets. Further still, a calibrationfunctionality is enabled in that the utilization of multiplephotometers/photometer channels to detect/measure the same target infurtherance of comparative assessment of the findings, with correctionmethods, thereafter and as the circumstances warrant, applied such thateach photometer “reports” the same signal for the same target. Yetfurther, multiple scanning rails may be provided such that an array tapeor the like feeds continuously through all scanning modules forsimultaneous, parallel scanning optical readers for differentfluorophores.

Thus, since the structures and/or methods of the scanning photometerdisclosed herein may be embodied in other specific forms withoutdeparting from the spirit or general characteristics thereof, some ofwhich forms have been indicated, the embodiments described and depictedherein/with are to be considered in all respects illustrative and notrestrictive. Accordingly, the scope of the subject invention is asdefined in the language of the appended claims, and includes notinsubstantial equivalents thereto.

1. A photometer comprising: a. a first fluorophore excitation source; b.a second fluorophore excitation source; c. an objective lens,characterized by an optical path, through which excitation energy fromsaid first and said second fluorophore excitation sources pass; and, d.a first emission detector for detecting both a first fluorophoreemission originating from excitation of a first fluorophore and a secondfluorophore emission originating from excitation of a secondfluorophore.
 2. The photometer of claim 1 operatively supported by ascanning rail carriage for reversible travel.
 3. The photometer of claim1 further comprising a third fluorophore excitation source, excitationenergy from said third fluorophore excitation source passing throughsaid objective lens.
 4. The photometer of claim 1 further comprising athird fluorophore excitation source and a second emission detector fordetecting a third fluorophore emission originating from excitation of athird fluorophore.
 5. The photometer of claim 1 further comprisingexcitation energy conditioning elements operatively interposed betweensaid first and second excitation sources and said objective lens.
 6. Thephotometer of claim 5 wherein said excitation energy conditioningelements comprise excitation filters and beam splitters for selectpassage of excitation energy to and along said optical path of saidobjective lens.
 7. The photometer of claim 5 further comprising emissionenergy conditioning elements operatively interposed between saidobjective lens and said first emission detector.
 8. The photometer ofclaim 7 wherein said emission energy conditioning elements comprise beamsplitters and an emission filter for select passage of emission energyto said first emission detector.
 9. The photometer of claim 1 furthercomprising a first beam splitter, excitation energy from one of saidfirst fluorophore excitation source and said second fluorophoreexcitation source being reflected thereby.
 10. The photometer of claim 9further comprising a second beam splitter, excitation energy from bothof said first fluorophore excitation source and said second fluorophoreexcitation source being reflected thereby.
 11. The photometer of claim 1further comprising emission energy conditioning elements operativelyinterposed between said objective lens and said first emission detector.12. A photometric scanning method comprising: a. passing an objectivelens across a series of spaced apart targets; b. transmitting energyfrom a first fluorophore excitation source through an optical path ofsaid objective lens for receipt by a target of said series of spacedapart targets in furtherance of exciting a first fluorophore of saidtarget; and, c. transmitting energy from a second fluorophore excitationsource through said optical path of said objective lens for receipt bysaid target of said series of spaced apart targets in furtherance ofexciting a second fluorophore of said target.
 13. The photometricscanning method of claim 12 wherein energy transmission from said firstfluorophore excitation source is simultaneous with energy transmissionfrom said second fluorophore excitation source.
 14. The photometricscanning method of claim 12 wherein energy transmission from one of saidfirst fluorophore excitation source and said second fluorophoreexcitation source is periodic.
 15. The photometric scanning method ofclaim 12 wherein energy transmission from one of said first fluorophoreexcitation source and said second fluorophore excitation source isalternated with energy transmission of another of said first fluorophoreexcitation source and said second fluorophore excitation source.
 16. Thephotometric scanning method of claim 12 wherein energy transmission fromone of said first fluorophore excitation source and said secondfluorophore excitation source is pulsed during energy transmission ofanother of said first fluorophore excitation source and said secondfluorophore excitation source.
 17. The photometric scanning method ofclaim 12 wherein excitation of said first and second fluorophores issimultaneous.
 18. The photometric scanning method of claim 12 whereinexcitation of said first and second fluorophores is alternated.
 19. Thephotometric scanning method of claim 12 wherein said passing saidobjective lens across a series of spaced apart targets is characterizedby a constant velocity.
 20. The photometric scanning method of claim 12further comprising detecting, via a single emission detector, first andsecond fluorophore emissions corresponding to an excitation of first andsecond fluorophores of said target.
 21. The photometric scanning methodof claim 12 further comprising transmitting, through said optical pathof said objective lens, first and second fluorophore emissionscorresponding to an excitation of first and second fluorophores of saidtarget.
 22. The photometric scanning method of claim 12 wherein saidseries of spaced apart targets are embodied in an array tape.
 23. Thephotometric scanning method of claim 12 wherein said series of spacedapart targets are embodied in a plate.
 24. The photometric scanningmethod of claim 12 wherein said series of spaced apart targets comprisecolumns of targets.
 25. The photometric scanning method of claim 24wherein said objective lens passes across a column of columns of targetsin a first direction, and thereafter passes across a column of targetsadjacent said column in a direction reverse of said first direction.